Isotopically labeled chemically stable reagents and process for the synthesis thereof

ABSTRACT

A radioisotope labeled reagent includes a compound having the general formula (I),
 
L-( a C b H 2 ) n   a C 5b H 3   (I)
 
where a in each occurrence independently is a carbon mass number between 11 and 14 inclusive, b in each occurrence independently is a hydrogen mass number between 1 and 3 inclusive, such that a in each occurrence is not 12 simultaneously with b in each occurrence being 1; L is a leaving group R 1 SO 2 —O—, R 1 —S—,  12 C 1 H 3 ( 12 C 1 H 2 ) n —S—R 1 C(O)O—, NC—, (R 1 ) 3 P—, XMg— and Li—, where n is an integer between 0 and 3 inclusive, where X is chloro, bromo or iodine, where R 1  is H, aryl, a substituent containing aryl, C 1 -C 20  alkyl, a substituent containing C 1 -C 20  alkyl, C 2 -C 20  alkenyl, a substituent containing C 2 -C 20  alkenyl, C 2 -C 20  alkynyl, and a substitute containing C 2 -C 20  alkynyl with the proviso that when n is 0, a is 13 and b is 2 and R 1  in R 1 —S is not aryl.

RELATED APPLICATION

This application is a continuation of the U.S. patent application Ser.No. 10/559,047 filed Mar. 28, 2006, which is the United States nationalstage of PCT Application No: PCT/US2004/016898 filed May 28, 2004 andclaims priority of U.S. Provisional Patent Application Ser. No.60/320,238 filed May 30, 2003, each of which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates in general to reagents having isotopiclabels and in particular to tritiated reagents having greater stabilitythan alkyl halides.

BACKGROUND OF THE INVENTION

Alkyl halides are versatile alkylating agents in organic chemistry.Methyl halides are particularly popular as alkylating agents.Representative of the methyl halides is methyl iodide, which in a purestate is a clear liquid that over time becomes brown as a result ofdecomposition to form various iodine-containing species. Methyl iodideis often stabilized through the addition of a solid metal such asmercury or copper to the storage vessel. As alkyl halides includingmethyl iodide are susceptible to actinic degradation and free radicaldecomposition, alkyl halide storage is often problematic. Nonetheless,aged alkyl halides are readily restored to usable form through adistillation process.

The handling of isotopically enriched alkyl halides is made all the moredifficult by radioisotope emissions creating free radicals that speedthe chemical decomposition of the alkyl halide. Distillation to purifyusable alkyl halides from a decomposing isotopically enriched alkylhalide is both technically challenging to perform and highly wasteful ofradioisotopes.

Owing to the complexities of handling radioisotopes, isotopicallylabeled reagents tend to be small molecules that can be synthesized andused quickly. [Methyl-³H]methyl iodide is a common methylating reagentused in the synthesis of methyl-labeled radiochemicals. Unfortunately,the rapid degradation of tritiated methyl iodide and otherisotope-enriched alkyl halides means that these reagents must be usedrapidly after synthesis. The requirement of rapid usage of isotopicallylabeled alkyl halides entails a scheduled batch production of thereagent followed by numerous reagent consumptive reactions beingperformed thereafter. The net result is that labeling reactions cannotbe efficiently performed but instead are tied to the schedule of alkylhalide production. Additionally, an excess of isotopically enrichedalkyl halide is necessarily produced to preclude the possibility ofperforming a second batch production to account for any shortfall. Theresulting excess production of isotopically enriched alkyl halide iswasteful of materials and increases the waste disposal volume. Thus,there exists a need for an isotopically enriched alkylating reagent thathas a longer shelf life than the corresponding alkyl halide without lossof specific isotope activity.

SUMMARY OF THE INVENTION

A radioisotope labeled reagent includes a compound having the generalformula (I),L-(^(a)C^(b)H₂)_(n) ^(a)C^(b)H₃  (I)where a in each occurrence independently is a carbon mass number between11 and 14 inclusive, b in each occurrence independently is a hydrogenmass number between 1 and 3 inclusive, such that a in each occurrence isnot 12 simultaneously with b in each occurrence being 1; L is a leavinggroup R¹SO₂—O—, R¹—S—, ¹²C¹H₃(¹²C¹H₂)_(n)—S—R¹C(O)O—, NC—, (R¹)₃P—, XMg—and Li—, where n is an integer between 0 and 3 inclusive, where X ischloro, bromo or iodine, where R¹ is H, aryl, a substituent containingaryl, C₁-C₂₀ alkyl, a substituent containing C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, a substituent containing C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, and asubstitute containing C₂-C₂₀ alkynyl with the proviso that when n is 0,a is 13 and b is 2 and R¹ in R¹—S is not aryl.

A process for preparing a compound of Formula I includes reacting anisotope enriched methyl halide, where L is a leaving grouprepresentative of ^(a)C^(b)H₃(^(a)C^(b)H₂)_(n)X with [L]^(y−)M_(p) ^(Z+)or Mg, M is a metal ion or onium ion, Z+ is a cationic valency of M, Y−is an anionic valency of L, p is the absolute value of the anionicvalency divided by the cation valency; preferably under anhydrousconditions in an aprotic solvent. Protic solvent and small amounts ofwater are tolerated in certain synthetic schemes.

A method of isotopically alkylating a target molecule involves mixingthe target molecule under reaction conditions with an effective amountof compound according to Formula I. The compound of Formula I is usefulin isotopically labeling a target molecule and has the advantage ofextended storage stability relative to the corresponding methyl iodidereagent.

A commercial package includes a compound of Formula I together withinstructions for the use thereof as an isotopic labeling reagent. Theenhanced chemical stability of a compound of Formula I affords thepossibility of performing isotopic labeling reactions remote from thereagent synthesis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has utility as an isotopic labeling reagent havingsuperior storage properties as compared to the corresponding alkylhalide. The labeling reagents according to the present invention areoperative to introduce carbon-11 [¹¹C], carbon-12 [¹²C], carbon-13 [¹³C]and carbon-14 [¹⁴C]. Independent of whether the methyl carbon is aradioisotope, the three hydrogen atoms making up a methyl group are 3hydrogen-1 [¹H₃], 3 hydrogen-2 [²H₃] or 3 hydrogen-3 [³H₃] with thecondition that at least one of the carbon or the three hydrogens of themethyl group are naturally occurring minor constituent isotopes.Preferably, methylene (—^(a)C^(b)H₂—) groups in higher alkyls share theisotopic identity of the thermal methyl group. As used herein, deuteriumis appreciated to be synonymous with hydrogen-2 and tritium synonymouswith ³H. While according to the present invention all three hydrogenatoms that compose the methyl group are isotopically identical, it isappreciated that isotopically mixed hydrogen atoms are operative to forma methyl group. An isotope labeled reagent is a compound having ageneral formulaL-(^(a)C^(b)H₂)_(n) ^(a)C^(b)H₃  (I)where a in each occurrence independently is a carbon atom mass number ofbetween 11 and 14 inclusive, b in each occurrence independently is ahydrogen atom mass number of between 1 and 3 inclusive, and at least oneof a and b is a naturally occurring minor isotope constituent. An“isotope” for carbon is defined herein to include instances where themajority of carbon atoms have a carbon atomic mass number of other thancarbon-12. An enriched isotope of hydrogen has as the majority hydrogenatomic mass number a value of 2 or 3. Preferably, the carbon atomic massnumber is 12 and all the hydrogen atomic mass numbers are 3.

The leaving group L is selected to represent a chemically stable leavinggroup upon reaction with a target molecule nucleophile. The leavinggroup L is representative of: R¹SO₂—O—, R¹—S—, ¹²C¹H₃(¹²C¹H₂)_(n)—S—,R¹C(O)O—, NC—, (R¹)₃P—, XMg— and Li—. R¹ is hydrogen, aryl, asubstituent containing aryl, C₁-C₂₀ alkyl, a substituent containingC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, a substituent containing C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl, and a substituent containing C₂-C₂₀ alkynyl. It isappreciated that the substituent, if present, is non-reactive towardsintramolecular reaction within the compound. An aryl group according tothe present invention is a monovalent monocyclic or bicyclic aromatichydrocarbon radical of 6 to 10 ring atoms and illustratively includesnaphthyl, a substituent containing naphthyl, phenyl, and a substituentcontaining phenyl with the proviso that a is not 13 and b is not 2 inthe instance when the leaving group L is the mercapto aryl R¹—S—. C₁-C₂₀alkyl, C₂-C₂₀ alkenyl and C₂-C₂₀ alkynyl leaving groups operative hereininclude linear, branched, cyclic and bicyclic species. A substituentoperative herein to modify an aryl, alkyl, alkenyl, or alkynyl replacesa hydrogen bonded to a carbon atom with each substituent independentlybeing selected from alkyl, amino, cycloalkyl, halo, nitro, cyano, —OR²,acyl, and —COOR³. Alkyl substituents are C₁-C₆ and preferably, C₁-C₄.Operative alkyl substituents illustratively include methyl, ethyl,n-propyl, isopropyl, butyl, isobutyl, terbutyl, pentyl, isopentyl, andhexyl. A cycloalkyl substituent operative herein is a C₅ or C₆cyclopentyl or cyclohexyl species. A hetero cycloalkyl operative hereinis selected from furanyl, tetrahydrofuranyl, epoxi, tetrahydropyranyl,dioxynyl, thiacyclopentyl, azeridyl pyrolidyl, piperadyl, morpholyl, andalkyl substituted forms thereof. Halo substituents operative herein areselected from fluoro, chloro, bromo and iodo. A substituent amino groupis selected from NH₂, NHRR⁴ or NR⁴R⁵. R² is selected from hydrogen,C₁-C₆ alkyl, C₅ or C₆ cycloalkyl, a heterocycloalkyl as described above,or a substituent containing phenyl. R³ is hydrogen or C₁-C₆ alkyl. R⁴and R⁵ in each occurrence are independently selected from C₁-C₆ alkyl,aryl as described above, C₁-C₆ alkoxy, and C₆ phenoxy.

Specific examples of isotopically labeled reagents according to thepresent invention include methyl sulfonic acid, methyl tosylate, methylmesylate, methyl nosylate, dimethyl thioether, terbutyl methylthioether, methyl benzoate, methilide triphenyl phosphene, methylmagnesium chloride, and methyl lithium. Additionally, it is appreciatedthat substituents of a leaving group L optionally incorporate a dyemoiety illustratively including cyanine, rhodamine or other conjugatedaromatic functionality to render inventive reagent an isotopic, as wellas a spectroscopic labeling compound. It is further appreciated thateach of the reagent compounds produced according to the presentinvention, while having superior stability and handling properties ascompared to isotopically labeled methyl halides, has limitations as tothe reactions in which it is operative. By way of example, methylmagnesium halides and methyl lithium are operative in aqueousenvironments only to form isotopically labeled methane whereas inanhydrous environments are suitable to perform labeling reactions wellknown to the art. Methylide phosphenes are operative in performingWittig reactions, while in general inventive compounds are useful inperforming nucleophilic substitution labeling reactions.

A process for preparation of a compound of Formula I is summarized bythe following reaction:

where L is the leaving group described with respect to the compound ofFormula I with the exclusion of magnesium which does not react by ametathesis reaction but instead is additive to form the resulting methylGrignard reagent. And in the case of methyl lithium, the reaction IIA issatisfied by [L]^(Y−) being Li¹⁻. M is a metal ion or onium ion. M is alithium ion, sodium ion, potassium ion, magnesium ion, calcium ion,barium ion, cesium ion, silver ion, zinc ion, copper ion, cobalt ion,iron ion, nickel ion, manganese ion, titanium ion, lead ion, chromiumion, vanadium ion, ruthenium ion, yttrium ion, lanthanoid ion, actinoidion, tetrabutylammonium ion, tetraethylammonium ion, tetramethylammoniumion, triethylmethylammonium ion, triethylammonium ion, pyridinium ion,imidazolium ion, hydrogen ion, tetraethylphosphonium ion,tetramethylphosphonium ion, tetraphenylphosphonium ion,triphenylsulfonium ion, or triethylsulfonium ion. Preferably, the metalion M is selected to produce a stable metal halide that facilitatesseparation of the inventive compound of Formula I therefrom. Preferredmetal ions, include silver and other transition metals. X as per thecompound of Formula I is chloride, bromide or iodide. As shown in thegeneral Formula IIA, the valency of M^(Z+) cation is preferably from 1to 3, inclusive. In instances where the valency of M^(Z+) is greaterthan 3, dissolution of ionic metal complex in solvent tends to occur asa result of increases in crystal lattice energy. As a result, in a morepreferred embodiment, the valency of M^(Z+) is 1. As shown in thegeneral Formula IIA, the valency Y− of L is similarly preferably from 1to 3 with a valency of 1− being most preferred. P is the absolute valueof the anionic valency to the Y− divided by the cation valency Z+ andthereby provide net charge neutrality.

Methyl iodide for use in reaction Formulas IIA and IIB are produced byestablished techniques. ¹²C³H₃I is produced by a well-establishedtechnique. Dass, Desmond V.; Dempsey, Victor J.; Martin, R. Wayne;Odell, Allan L., Journal of Labelled Compounds and Radiopharmaceuticals(1987), 24(5), 517-20; Liu, Yu-Ying; Chen, Journal of Labelled Compounds& Radiopharmaceuticals (1996), 38(1), 71-6, and Schwob, R.; Wuersch, J.,Journal of Labelled Compounds and Radiopharmaceuticals (1978), 14(3),355-60. ¹¹C¹H₃ methyl iodide is produced with the well-establishedreaction of carbon-11 dioxide with lithium aluminum hydride andsubsequent hydrolysis with hydroiodic acid. ¹³C with 1, 2 or 3 deuteriumatoms present in the methyl group are produced as detailed in U.S. Pat.No. 6,713,044 B2. Powdered magnesium and reagents of the form[L]^(Y−)M^(C+) _(p) according to the present invention are conventionalto the art and in most cases commercially available reagents. Thereaction conditions to perform the reaction of Formulas IIA and IIB inorder to produce a compound according to Formula I are known to the artfor a specific reaction involving an alkyl halide and are in generalcharacterized by reaction in an aprotic solvent under anhydrousconditions. Further guidance as to reaction conditions is found withreference to Grignard reagent synthesis and the Williamson ethersynthesis. The reaction process of Formulas IIA and IIB yield a productthat has greater chemical stability than the corresponding alkyl halidewhile preserving the isotopic character of the alkyl halide. Theresulting inventive reagents are further characterized by beingnonvolatile and of lesser toxicity than the corresponding alkyl halide.

A commercial package according to the present invention includes acompound of Formula I, preferably in purified form, together withinstructions for the use of the compound as an isotopic labelingreagent. One of skill in the art will appreciate that those compounds ofFormula I that represent esters of strong acids are well suited asreagents for labeling nucleophiles by way of an S_(N)2 reactionmechanism. Alternatively, Grignard reagents and alkyl lithium reagentsare well suited for the production of ketones from carboxylic acids andcarboxylic acid derivatives illustratively including amides and esters.In the case of esters, it is appreciated that the resulting ketone inthe presence of a Grignard reagent is unstable resulting in a methylatedtertiary alcohol.

The invention is better understood with respect to the followingexamples. These examples are given as being illustrative of the presentinvention and are not to be construed as limiting the invention eitherin spirit or in scope as many modifications both in materials andmethods will be apparent to those skilled in the art upon reading thesame. While the following examples all pertain to ¹²C³H₃I as a startingmaterial, it is appreciated that other isotopically enriched alkylhalides according to the present invention are equally operative herein.

EXAMPLE 1 Typical preparation of [methyl-¹²C³H] methylpara-toluenesulfonate (III)

0.4 mmol (35 Ci, carrier-free) of ¹²C³H₃I is sealed into a glassreaction bulb with silver tosylate (140 mg, 0.5 mmol) and 5 ml ofanhydrous acetonitrile. The reaction is heated to 80° C. overnight.Labels are removed, and the residue dissolves in ethyl acetate. Theyield is 30 Ci (85%) of [Methyl-¹²C³H]methyl para-toluenesulfonate(III). The labeled material and authentic cold standard comigrated onthin layer chromatography (TLC), (Whatman LK6DF, hexane-ethyl acetate,10:3, R_(f)=0.5). Stored at 600 mCi/ml in ethyl acetate at 25° C., theradiochemical purity as determined by TLC as above is unchanged after 20days.

EXAMPLE 2 Synthesis of L-[N-methyl-¹²C³H] quinuclidinyl benzilate methylchloride (IV) with [methyl-¹²C³H]methyl para-toluenesulfonate (III)

600 mCi (0.0073 mmol) of [methyl-¹²C³H]methyl para-toluenesulfonate(III) and 10 mg (0.03 mmol) of R-(−)-3-quinuclidinyl benzilate arestirred in 2 ml of methanol at room temperature overnight. TLC of thereaction (silica gel GHLF, n-butanol-acetic acid-water, 4:1:1) showsonly product and unreacted tosylate. The whole is purified on HPLC(Zorbax SB-C8, methanol-1% TEAA pH4, gradient) to give after addition ofa chloride source L-[N-methyl-¹²C³H] quinuclidinyl benzilate methylchloride (IV). The specific activity is determined to be 82.0 Ci/mmol bymass spectral analysis, and the radiochemical purity determined by HPLCas above is 99%.

EXAMPLE 3 Typical preparation of [methyl-³H]methylpara-nitrobenzenesulfonate (V)

0.14 mmol (12 Ci, carrier-free) of ¹²C³H₃I is sealed into a glassreaction bulb with silver nosylate (62 mg, 0.2 mmol) and 5 ml ofanhydrous acetonitrile. The reaction is heated to 80° C. overnight.Labels are removed, and the residue dissolves in ethyl acetate. Theyield is 6.16 Ci (51%) of [methyl-¹²C³H]methylpara-nitrobenzenesulfonate (V). The labeled material and authentic coldstandard comigrated on TLC (Whatman LK6DF, hexane-ethyl acetate, 10:3,R_(f)=0.5). Stored at 28.4 mCi/ml in hexane-ethyl acetate (8:2) at 25°C., the radiochemical purity as determined by TLC as above is unchangedafter 4 months.

EXAMPLE 4 Synthesis of [methyl ester-³H]carfentanil (VI) with[methyl-¹²C³H ]methyl para-nitrobenzenesulfonate (V)

400 mCi (0.005 mmol) of [methyl-³H]methyl para-nitrobenzenesulfonate (V)and 1.5 mg (0.0036 mmol) of carfentanil sodium salt are stirred in 0.2ml of anhydrous DMF at room temperature overnight. TLC of the reaction(Whatman LK6DF, chloroform-methanol-ammonium hydroxide, 100:2:1) showonly product and unreacted nosylate. Analysis by HPLC on ODS show that91% of the activity coeluted with cold standard. A portion is purifiedon HPLC (Zorbax SB-C18, acetonitrile-0.1% trifluoroacetic acid,gradient) to give [Methyl ester-²C³H]carfentanil (VI). The specificactivity is determined to be 80.0 Ci/mmol by mass spectral analysis, andthe radiochemical purity determined by HPLC as above is 99%.

EXAMPLE 5 Preparation of [Methyl-³H]-Raclopride (VII)

Raclopride is prepared at 80.5 Ci/mmol by heating the reaction to 70° C.in DMSO. The methyl nosylate (V) is able to be dispensed by volume, andthe solvent removed to leave the reagent ready for use in the reactionvessel. In the methylation of the raclopride precursor, thestoichiometry of the reaction is able to be carefully controlled tominimize dimethylation.

EXAMPLE 6 Methylating Comparison C³H₃I and Methyl Nosylate (V)

The methylating ability of methyl iodide vs. methyl nosylate is comparedin a competition experiment. The potassium salt of 2-naphthylacetic acidis stirred in dimethyl formamide with one equivalent of cold methyliodide and one equivalent of tritiated methyl nosylate (V). The purifiedmaterial is determined to be 86 Ci/mmol. In this experiment, thenucleophile had been preferentially methylated by the tritiated methylnosylate (V) with only a small fraction reacting instead with theunlabeled methyl iodide.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual application orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. An isotopically labeled reagent comprising: a compound having aformulaL-(^(a)C^(b)H₂)_(n) ^(a)C^(b)H₃  (I) where a in each occurrence isindependently a carbon atomic mass number of between 11 and 14 inclusiveand wherein said compound is isotopically enriched with at least onecarbon where a is 11, 13 or 14, where b in every occurrence is ahydrogen atomic mass number of between 1 and 3 inclusive, where L is aleaving group R¹SO₂—O—, where n is 0, wherein R¹ is para-NO₂—C₆H₄. 2.The reagent of claim 1 wherein a is
 14. 3. A process for preparing acompound of Formula I according to claim 1 comprising the step ofreacting ^(a)C^(b)H₃(^(a)C^(b)H₂)_(n)X with [L]^(Y−)M_(p) ^(Z+), where Mis a metal ion or onium ion, Z+ is a cationic valency of M, Y− is ananionic valency of L, P is the absolute value of the ionic valencydivided by the cationic valency; and where the reaction occurs underanhydrous conditions in an aprotic solvent.
 4. The process of claim 3wherein ^(a)C^(b)H₃ (^(a)C^(b)H₂)_(n)X is reacted with [L]^(y−)M_(p)^(Z+) reagent.
 5. The process of claim 3 wherein the step of reactingoccurs with heating to a temperature less than or equal to a boilingtemperature for said aprotic solvent.
 6. The process of claim 3 wherein^(a)C^(b)H₃ (^(a)C^(b)H₂)_(n)X is reacted with ¹²C³H₃I.
 7. A method ofisotopically alkylating a target molecule comprising mixing underreaction conditions with said target molecule an effective amount of acompound of Formula I according to claim
 1. 8. The method of claim 7wherein said target molecule is a nucleophile.
 9. The method of claim 7wherein said target molecule is a metallic salt of an organic anion. 10.The method of claim 7 wherein said target molecule is a tertiary amine.11. A commercial package comprising a compound of Formula I according toclaim 1 together with instructions for the use thereof as an isotopiclabel alkylating reagent.
 12. An isotopically labeled reagentcomprising: a compound having a formulaR¹—SO₂O—^(a)C^(b)H₃ wherein said compound is isotopically enriched wherea in each occurrence is independently a carbon atomic mass number of 11,13, or 14, where b in each occurrence is independently a hydrogen atomicmass number of between 1 and 3 inclusive, where R¹ is a substituentcontaining aryl where the substituent is selected from the groupconsisting of: C₂-C₆ alkyl, amino, cycloalkyl, halo, nitro, cyano, —OR²,acyl, and —COOR³, and wherein b is 3 in all occurrences.